& Prevention Prevalence of Human Papillomavirus Types in ...cebp.aacrjournals.org/content/cebp/19/9/2389.full.pdf · Prevalence of Human Papillomavirus Types in High-Grade Cervical

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valence of Human Papillomavirus Types in High-Grade

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vical Intraepithelial Neoplasia and Cancer in Italy

esca M. Carozzi1, Maria L. Tornesello2, Elena Burroni1, Giovanna Loquercio2, Giuseppe Carillo3,
io Angeloni4, Aurora Scalisi5, Rosalba Macis6, Francesco Chini7, Franco M. Buonaguro2, and Giorgi Rossi7; for the HPV Prevalence Italian Working Group
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kground: The aim of this multicentric study was to identify human papillomavirus (HPV) type distri-in invasive cervical cancer and high-grade cervical intraepithelial neoplasia 2/3 (CIN2/3) in Italy.

thods: Cases were sampled through the electronic databases at the pathology units of eight centers in sixs from central and southern Italy. HPV types were detected from paraffin-embedded tissue samples andal specimens through amplification of HPV DNA with GP5+/GP6+ primers, followed by genotypingeverse line blot (RLB). Untyped HPV-positive samples were sequenced. HPV-negative samples under-ested PCR, followed by either RLB or sequencing. Finally, the remaining HPV-negative samples were

fied with primers targeting the virus E6 to E7 regions.ults: From 1,162 cases initially selected, 722 samples were further analyzed: 144 CIN2, 385 CIN3, 157ve squamous carcinomas, and 36 adenocarcinomas. Samples (6.9%) were HPV negative. The proportionV16/18 was 60.8%, 76.6%, and 78.8% in CIN2, CIN3, and invasive cancers, respectively (P trend =. There was a significant decreasing trend of HPV16/18 with age in invasive cancers, going fromn women <35 years to 73% in women >55 years (P = 0.036). The proportion of coinfections was, 15.5%, and 10.0% in CIN2, CIN3, and invasive cancers, respectively (P trend = 0.07).clusions: The proportion of invasive cancers caused by HPV16/18 decreases with age at diagnosis.act: The absolute risk of an invasive cancer due to non-HPV16/18 in women under 35 is extremely low.
Imp
This finding might prompt us to rise the age at which public HPV screening for vaccinated women shouldstart. Cancer Epidemiol Biomarkers Prev; 19(9); 2389–400. ©2010 AACR.

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new approaches for the prevention of cervical can-ve emerged over the last few years: vaccinationst human papillomavirus (HPV) in adolescent girlsPV test as primary screening test (1-8). In Italy, thee is currently recommended for all 11-year-old girlse vaccination campaign aims at covering 95% of thepopulation within 5 years since it began in 2008 (9).ulation-based studies of HPV genotype prevalenceeded to predict how these two approaches mightnce cervical cancer prevention. To monitor whether

ccination might induce changes in the dis-V types in high-grade lesions or cancer,

distribvariatdecisiin invcan hfor vaSev

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ns: 1ISPO, Florence, Italy; 2National Cancer Instituteples, Italy; 3ASL Caserta 2, Caserta, Italy; 4ASLItaly; 5ASL Catania, Catania, Italy; 6ASL Cagliari,7Laziosanità, Rome, Italy

ary data for this article are available at Cancer Epide-rs & Prevention Online (http://cebp.aacrjournals.org/).

uthor: Paolo Giorgi Rossi, Laziosanità, Via di Santae 00198, Italy. Phone: 39-06-83060438; Fax: [email protected]

9965.EPI-10-0131

ssociation for Cancer Research.

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on February 15, 2019. © 2cebp.aacrjournals.org from

ed solid baseline data on HPV epidemiology notn the healthy population but also in women withal neoplasia. Moreover, the ability to develop theappropriate screening algorithm and protocols fornated cohorts depends on our understanding ofsolute risk of non-HPV16/18 cervical cancer by

nd history of screening.eta-analysis of worldwide studies showed that thertion of HPV16 and HPV18 among invasive can-s quite stable and that they are present in the vastity of invasive cases without any correlation withographic location (10, 11). On the other hand, theution of other HPV types shows some geographicion (12-15). Useful information in public healthon making is the proportion of non-HPV16/18asive cancers from younger women. Knowing thiselp to determine the best age at which screeningccinated women should start (16).eral studies about HPV types in precancer and can-ve been conducted in Italy, but in most of them,le size was small. Table 1 summarizes results andle sizes from previous Italian studies.aim of this multicentric study was to describe the
ution of HPV types in cervical neoplasia, by age,of lesion, and histologic type, with an appropriate
2389

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-SIL 73.3 18 9 25- 64 ASCU S/AGUS 40.7 45% 5% 50% i
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reviations: L-SIL, low-grade SIL; n.r., not reported.mber of HPV+ samples.


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le size, from relatively recent samples collected bylaboratories in six different regions from centralouthern Italy. Our results might have an effect onons about the age at which HPV screening forated women should start.

rials and Methods

tion of casess multicentric study involved eight centers fromifferent Italian regions: Abruzzo (Atri), Lazioiovanni Hospital in Rome and Belcolle Hospitalerbo), Campania (Naples: National Cancer Insti-ond Pascale), Sicily (Catania), Sardinia (Cagliari),uscany (Florence: ISPO and S. Maria Annunziataital). Specimens were retrieved from the archivescenters, but molecular analyses were centralizedo laboratories (ISPO in Florence and Nationalr Institute Fond Pascale in Naples).coordinating laboratory collected the lists of pa-with cervical neoplasia from the databases of theipating centers' pathology units (list of ICD-O, SNOWMED, text). Information collected includednd place of birth, date of diagnosis, diagnosis code,t. The calendar period we considered coincidedhe availability of electronic archives (1999-2008).y histologically confirmed diagnoses were includedstudy. All invasive squamous cancers, adenocarci-(in situ or invasive), and all cases from women

abroad were included in the study, whereas a ran-ample of cervical intraepithelial neoplasia 2 (CIN2)IN3 was drawn to reach a minimum of 1,200 cases.ists of sampled women were then given back tocenter to confirm the original diagnosis checkingmedical records. Finally, the pathology units re-

d the samples from their specimen bank, selectingecimen with the most severe diagnosis. When mul-araffin-embedded tissues were present for a singlehe tissue sample used for this analysis was selectedfollowing order of priority: the original cervicalbiopsy sample, followed by the loop excision

men, followed by the hysterectomy specimen.logic samples were not available for some screen-ted lesions (157 CIN2/3 and 1 invasive cervicaloma), so HPV typing was done on the originalal samples.

e samplesaffin-embedded tissue samples were subjected to aich technique for which we obtained an initialtissue section for H&E staining, followed byfive 10-μm sections that were collected in twoate tubes for PCR, and immediately sent to thence facility.-safe precautionswere taken tominimize the risk ofmination during tissue processing. Sections were
om the tissue blocks with a standard microtome,lly cleaning the microtome and using a new part
in eacdurin

Cacrjournals.org

on February 15, 2019. © 2cebp.aacrjournals.org nloaded from

blade for each block, changing gloves betweenens, and using disposable toothpicks (sticks) to

er tissue ribbons to their storage tubes. Finally, tissuessing andHPV testingwere done indifferent facilities.each sample, the following information wased: date of sampling, type of material (biopsy orry sample), number of sections in each tube, andent.

logic evaluationE slides immediately adjacent to the tissue ribbonsor HPVDNA typing had to be reviewed by the locallogist for the presence of lesions to check that neo-c tissue had been included. In one center (Naples), thishologic check showed that the mean percentage ofcell staining was 64% (range, 5-100%), with a rele-roportion of sample slices containing a limited quan-neoplastic tissue within a healthy tissue context.

ogic samplesvical scrape samples were collected in ThinPrep vialining PreservCyt transport medium (Cytyc Corp.)Specimen Transport Medium (Digene, Qiagen). Ate of diagnosis, 400 μL of STM samples were stored

0°C, whereas 1.5 mL of the ThinPrep vial werefuged (10 minutes at 3,000 rpm) and washed twice0 mL of 1× PBS. Pellets were resuspended in 2 mLnd stored at −80°C until DNA extraction.

ratory proceduresA extraction and HPV genotyping were centralizedo laboratories (Florence and Naples) using harmo-protocols. ISPO in Florence analyzed samples fromzzo, Lazio, Sicily, Sardinia, and Tuscany, whereasascale National Cancer Institute analyzed samplesCampania.A extraction. DNAwas extracted from two sections) of paraffin-embedded tissue using QIAamp DNATissue kit (Qiagen) according to the manufacturer'sction with slight modifications. Paraffin was dis-in 1 mL of xylene. Shaking at room temperatureto 30 minutes followed. Sections were pelleted byfugation (14,000 rpm for 5 minutes) and washedin 100% ethanol. Pellets were dried at room temper-(for 30 minutes) and then at 37°C (for 15 minutes).samples were resuspended in 180 μL ATL Buffer0 μL proteinase K, mixed, and then incubated atovernight (until complete lysis). Samples were thenated for 1 hour at 90°C to partially reverse formal-e modification of nucleic acids. After this step, thefacturer's protocol was strictly followed and DNAluted in 100 μL ATE Buffer.A from cervical specimens was extracted usingmp DNA Mini kit (Qiagen) according to the manu-er's instructions.ample known to be negative for HPV was included
h batch of extraction to exclude any contaminationg this phase.
ancer Epidemiol Biomarkers Prev; 19(9) September 2010 2391



ForspectrHP

ent PFor a(30) oHPVthe mAn H(a pufree sductsethidition. GHPVthe LAll saweretion o45, 51HPV(26, 5visualkit. Ation isof theterpreGP5

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(Continued on the following page)

Carozzi et al.

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each sample, DNA concentration was evaluated byophotometry and expressed in ng/μL.V typing. HPV genotyping was done using differ-CR-based strategies in a well-defined algorithm.ll samples, we initially used a PCR assay basedn GP5+/GP6+ primers (Consensus High RiskGenotyping kit, Digene, Qiagen), according toanufacturer's instruction with slight modifications.PV-positive control and two negative PCR controlsrified DNA sample negative for HPV and a DNA-ample) were included in each PCR run. PCR pro-were loaded onto 2% agarose gel, stained withum bromide, and visualized under UV illumina-P5+/GP6+ primers amplify a broad spectrum of

genotypes by targeting a 150-bp fragment within1 open reading frame of the HPV genome (31).mples and controls, independent of gel results,subjected to the reverse line blot (RLB) for detec-f 12 high-risk HPV types (16, 18, 31, 33, 35, 39,, 52, 56, 58, and 59), 1 probably carcinogenictype (68), and 5 possibly carcinogenic HPV types3, 66, 73, and 82; ref. 32). RLB strips were analyzedly using an interpretation grid supplied with thebiotinylated poly(dT) control for conjugate reac-present in each strip to ensure good performancetest and proper alignment of the strips on the in-tation sheet.+/GP6+ PCR-negative and RLB-negative samplesamplified for the β-globin gene sequence using-PC04 primers (268-bp amplicon length) to assessintegrity (33).vercome any false negatives due to inhibitors com-present in formalin-fixed, paraffin-embedded tis-

ll samples negative for HPV at this first step wered on 1:10, 1:50, or 1:100 dilution.vercome misclassification of the HPV genotype re-g from potentially degraded DNA in aging archivalin-embedded tissues, samples that still remainedive for HPV on diluted DNA were reamplified us-NO-LiPA HPV Genotyping Extra Amp kit (Innoge-
), targeting a 65-bp fragment within the virus L1reading frame (34-36). INNO-LiPA genotyping kit
the dperfor

r Epidemiol Biomarkers Prev; 19(9) September 2010


lso used on the GP5+/GP6+ PCR-positive butegative samples because it allows for detection ofV types (16, 18, 26 31, 33, 35, 39, 45, 51, 52, 53, 56,, 66, 68, 73, 82, 6, 11, 40, 43, 44, 54, 70, 69, 71, andhis step was done only in Florence. All clearly vis-nes should be scored by using the INNO-LiPA HPVtyping Extra Reading Card. The remaining untypedles (GP5+/GP6+ PCR positive and RLB negative or-LiPA negative) underwent direct Sanger sequenc-f the GP5+/GP6+ PCR product using BigDye 1.1nator chemistry (Applied Biosystems) and ABI310 Genetic Analyzer (Applied Biosystems) tofy the specific HPV types probably not includedDigene genotyping kit or in the INNO-LiPA kit.

products were sequenced in both directions, andsulting sequences were compared with knownsequences in public databases using BLAST. A se-e was considered a match if it had >90% nucleotiderity to an HPV sequence in GenBank.overcome negative results due to low copy numberV DNA, samples that remained HPV negativee β-globin positive) were further analyzed byd PCR using the PGMY09/11 and GP5+/GP6+r sets (37), followed in Florence by RLB hybrid-n (Consensus High Risk HPV Genotyping kit),viously described, whereas in Naples samples un-nt direct sequencing of the nested amplificationcts.he last step, the remaining HPV-negative samplesbin positive) were amplified using HPV-specificrs for the E6 to E7 regions of four high-risktypes (18, 16, 45, and 31; ref. 33) to overcomenegative results due to integration events affectingPV L1 gene, which is the target of all the PCR-tests we used. The PCR products were loaded ontoarose gel, stained with ethidium bromide, and vi-ed under UV illumination. Samples that presentedcific PCR fragment were considered positive forecific HPV type.r goal in combining test results was to maximize

2. Sample structure, exclusion
recoverin ological s
etection of HPV gmance of any one

Cancer Epidemio

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led ed ailable analyzed
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4 67 127 (Belcolle-S. Giovanni) 1 32 223 ia (Cagliari) 6 0 46 Catania) 8 0 23 ny (Florence SMA-ISPO) 8 29 179 nia (Naples, Pascale) 205 31 50 124
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matic reviewsearched Medline using the terms (“HPV” ORan” AND “Papillomavirus”)) AND (“type” ORg”) AND (“Italy” OR “Italian”) AND (“cervix”

cervical”) AND (“cancer” OR “neoplasia”). Thewas updated on December 15, 2009. We excludeddies targeted to the healthy general population andositive women, and not regarding cervical neopla-e included only studies of pathologic populations.tracted the total HPV positivity, the proportion of6 among isolated HPV, the proportion of HPV18,he proportion of cases with HPV16 or HPV18 byof pathologic finding: histologic classification (ifted) or cytologic classification (if histology wasported).

sisfollowing analyses were planned a priori in thedesign: proportion of HPV16/18 by age group

rade of lesion (CIN2, CIN3 including adenocarcino-situ, invasive squamous carcinoma or adenocarci-, test for trend), proportion of HPV16/18 incarcinomas (in situ and invasive), proportion of6/18 in women born abroad, and distribution oftypes by grade of lesion. Furthermore, we presentstribution of types by center and the distributionnocarcinomas (in situ and invasive).as impossible to calculate a priori the precision

46 274 111

er) of this study because we did not know how HPV,The

typingHPVThe

rank oof lesHPV1is follThe

or infidenc72-81

invasive or adenocarcinomas would be retrieved.

lts

r sample included 1,162 cases. For 278 of them, itpossible to obtain a biological sample mainly be-

the paraffin block had not been correctly stored orse the histologic sample had been completely slicedsed for the original diagnosis. One hundred andtwo additional cases were excluded because theosis was not confirmed (131 of 884 = 14.8%, mostly
reported as “Uterus NAS,” which resulted to beetrial adenocarcinomas at the examination of
cance(P = 0

Cacrjournals.org


al records) or because there was no human DNAsample (31 of 753 = 4.1%; Table 2).laboratory in Florence analyzed 598 samples (158gic samples and 440 tissue specimens) and the oneples analyzed 155 tissue specimens.lorence, of 598 samples, 4 samples were negative forDNA, 537 were genotyped directly with GP5+/6+25 were typed on diluted DNA with GP5+/GP6+0 by INNO-LiPA, 14 by nested PCR, 8 samples werenced and for 3 of them a type was identified, and 2samples were genotyped with type-specific E6/E7primers. Finally, 3 samples remained untyped.aples, 31 of 155 tissue biopsies (20%) were nega-human DNA determined by amplification of the

bin gene. These were not considered in the follow-nalyses; 47 of 124 samples were genotyped with/GP6+ RLB, directly or diluted, 13 were typed byPCR, 13 were typed by HPV16 E6/E7-specific pri-5 samples were sequenced but remained undeter-, and 46 samples remained HPV negative.erall, 50 (6.9%) cases were negative for HPV DNA, 4.7%, and 6.7% for CIN2, CIN3, and invasive can-spectively). Forty-six of these were clustered in one, which analyzed paraffin blocks from an archivey used for several investigations, and all of themined <20% of tumor cells as evaluated by H&Eng. Excluding this center, 99.3% of samples resultedve for HPV DNA. Eight cases were positive forbut it was not possible to determine the type.flowchart in Fig. 1 summarizes the results of HPVin the two laboratories, whereas Table 3 shows the

strains found by type of lesion.most frequent type was HPV16 in all classes. Thef the other HPV types differs according to the gradeion: in CIN2/3, HPV16 is followed by HPV31,8, HPV58, and HPV33; in invasive cancers, HPV16owed by HPV18, HPV45, HPV31, and HPV58.percentage of cases with HPV16 or HPV18, alonecoinfection with other types, was 63.6% [95% con-e interval (95% CI), 54-72] in CIN2, 76.5% (95% CI,) in CIN3, and 80.2% (95% CI, 73-85) in invasive

1 36 0

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cases with HPV16 and/or HPV18 not associatedother high-risk HPV types: 51.2% in CIN2, 66.7%3, and 75.1% in invasive cancers.distribution of cases among adenocarcinomas isr to the types observed in squamous invasive can-6, 45, 18, 31, 58, and 35), and the only difference istly lower proportion of HPV16 (P = 0.5, differencesignificant); the overall proportion of HPV16/18 is(95% CI, 54-85), including coinfections with otherrisk HPV, whereas the percentage decreases toif we exclude coinfections.proportion of HPV16/18 in invasive cancers

s a statistically significant decreasing trend withom 92% in women under 34 to 73% in women agesd older (P = 0.036, test for linear trend). In womenonly HPV16, HPV18, and HPV45 were found,as in older ages, other and rare types were alsot. The trend is not significant for CIN2 (P = 0.4),

here is no trend for CIN3 (Fig. 2). The percentage

Figure 1. Flow diagram of the typin

es with HPV16 or HPV18 varied significantlyg geographic regions in precancer [χ2(5) = 24.0,

tions.frequ



.0005 for CIN2; χ2(5) = 14.9, P < 0.01 for CIN3]ot in invasive cancers [χ2(5) = 6.7, P = 0.25 forive cancers; Table 4]. There was no difference inoportion of HPV16/18 in CIN2 or CIN3 in liquid-cytology (LBC) specimens compared with histo-specimens (63% in LBC and 68% in histologicles for CIN2, P = 0.53; 69% in LBC and 71% inogic samples for CIN3, P = 0.62).sidering only CIN2 and CIN3, coinfections werefrequent, even if not significantly, in cytologic) than in tissue samples (14.8%; P = 0.3). No differ-in coinfections were found by age neither in CIN2/in invasive cancer. In 10.2% of invasive cancers, weved a coinfection, whereas in CIN3, the rate wasand in CIN2 it was 17.4%; the decreasing trendsignificant (P = 0.15, adjusted for type of specimen).ections varied also among regions [χ2(5) = 10.6, P =with a higher proportion in Campania (25.0%) andny (20.5%). HPV16 was involved in 77% of coinfec-

edures and results.

The frequency of combinations seems to reflect theency of single virus strains in each type of lesion,

Cancer Epidemiology, Biomarkers & Prevention



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if sample size does not allow for detection ofm or small differences. Matrices of all the observedinations of types by grade of lesion are available(Supplementary Table S1A-D).re were 106 women who were born abroad. Thertion of HPV16/18 in women born abroad was(95% CI, 56-83) in CIN2, 64.6% (95% CI, 49-78) in, and 90.0% (95% CI, 55-99) in invasive cancers.figures do not differ from the ones observed inn born in Italy.found an invasive cancer, positive for HPV DNA,
ive to both typing methods, in which we obtained asequence of HPV67.
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44 85 57(y35 60 1.7 39 6.1 104 58 0.3 54 0.0 344 23 6.0 55 4.3 3755 2 1.4 33 8.6 74.i.l H− 19 3.2 18 4.7 13ty

66 4.5 67 3.0 0715 2.4 20 5.5 1316 3.2 38 0.4 66 5.0 19 5.2 13 2.5 6 1.6 23 2.5 8 2.2 24 3.3 17 4.6 102 1.7 5 1.4 06 5.0 13 3.6 11 0.8 3 0.8 04 3.3 6 1.6 1

12 9.9 20 5.5 51 0.8 1 0.3 11 0.8 7 1.9 10 0.0 1 0.3 11 0.8 2 0.5 10 0.0 3 0.8 03 0 10 0 1

ty 4 2.8 1 0.3 2

risk, not included in typing target.

Cacrjournals.org


matic reviewe hundred and ninety-three articles were foundbMed according to the searching criteria; 14 offulfilled the inclusion criteria. Five articles reported

ts for histologically confirmed invasive cancers, 22, and 27), six reported results for preinvasives according to histologic findings (17-24), six re-results only according to cytologic results (25-29),

ne by both classifications (17). Results are summa-in Table 1. In all studies, the percentage of HPV16sed with the grade of pathologic findings. The per-
e of HPV16 among CIN2 ranged from 29% to 53%igh-grade squamous intraepithelial lesions (H-SIL)
ified by grade
histotype of
urc

an

01

lesion

sa

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6

6.4 31.7 73.6 57.1 0

8.3 0

5.4 49.2 54.2 30.7 01.4 21.4 07.0 70.0 00.7 00.0 00.7 03.5 20.7 10.7 00.7 00.7 00.0 0

00

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cer Epidemiol Biomark

0 American Associa

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22

8.3 129.4 533.9 205.6 29

0.0 50

8.6 644.3 538.6 630.0 265.7 130.0 130.0 380.0 70.0 200.0 40.0 115.7 392.9 40.0 90.0 20.0 40.0 3

41

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IN3 amou eno otal
9.45.06.67.9

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search.

inom
inom
n % n % n % n % n %

1
3 1 3 7 )
4
1 3 2 2 4 1 4 2 1 2 3 1 1 2 1 1 1
4
2 5 1 1 1 4 2 1 8
PV+ 121 84.0 366 95.1 142 90.4 35 97.2 664 92.0
1
pe
5 2 7 1 7 2 6 4 6 1 1 1 1
2

ped
8 6 2 7 1 8 2 7 4 7 r 1 77 3.6 80 6.5 17 2.4 5 1.4 99 5.2
r 18 alone 62 51.2 244 66.7 112 78.9 21 60.0 439 66.1tiple infections 21 17.4 57 15.6 11 7.7 7 20.0 96 14.5

determined risk.

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a similar range], whereas the proportion of CIN3

Figure 2. Proportion of HPV16/18 types by

d from 42% to 56% and in invasive cancers from founddoesparisoas HPstudiHPV1et al.ies hapropoare, hrangeThe

of neoportsis higprevioslightstudieinvas80% ibeforebetweto ranOn

HPV1servednot betherevanceand inWe

o 74%.

ssion

expected, given the natural progression of the dis-the overall HPV positivity was high; the observedlence agrees with results from epidemiologic studiesrdinated by the IARC (overall HPV/DNA preva-92.5%; ref. 38) and with more recently publishedn which HPV positivity ranges from 86% to 94%, de-ng on the continent (39). It is generally accepted thatences in the percentage of HPV-negative samplesstudies can be explained by differences in the meth-sed to assess HPV DNA positivity, in the histopath-quality of samples, and in the type of specimens

zed (biopsies, surgical specimens, or fresh tissues).ation of the present study has to take into accountwas not possible to check the histology of the bio-l sample collected at some centers, and that the vastity of negative samples originated from one of theses: If we ignore data collected at this facility, only 1 ofncers resulted to be HPV negative. Similarly, onlyositive samples were not typed.observed distribution of HPV types in CIN2 andslightly differs from the distribution observed byrge IARC meta-analysis for H-SIL in Europe (10):cases, both HPV16 and HPV58 show a higher fre-y. The same happens in invasive carcinomas, wherend a higher proportion of HPV16, HPV45, and
8 and a lower frequency of HPV18, HPV31, and3 compared with the IARC analysis.
changscree



le 1 summarizes the results of the 11 Italian studiesin our systematic review. The limited sample size

not permit an analysis for each type. Only a com-n for the proportion of HPV16 can be done, where-V18 shows large random fluctuations and fewes clearly report the proportion of cases with6 or HPV18 infection. In general, except for Sideri(22) and Tornesello et al. (27), all the reviewed stud-ve a few invasive cancers. Our estimates for thertion of HPV16 in CIN2, CIN3, and invasive cancerowever, close to or little over the upper level of thes found in previous Italian literature.proportion of HPV16/18 increases with the gradeplasia in our data set as well as in all literature re-(14, 15). Furthermore, the variability among regionsher for precancer than for cancer, as observed inus meta-analyses (10). The observed proportion isly higher than what is observed in previous Italians for each of the three classes: CIN2, CIN3, andive cancers. In particular, we observed a rate ofn cancer samples, similar to the results reportedby Tornesello and colleagues (27). The differenceen our study and the previous one is largely duedom fluctuation.the contrary, the finding of a higher proportion of6/18 in Italian studies compared with that ob-in the large IARC meta-analysis for Europe mayrandom. From cancer registry data, we know thatis a time trend for higher proportion of less ad-d cancers (40) but also for more screen-detectedterval cancers (41, 42).can envisage three possible explanations: (a) a real

of cervical lesion and age.

e has occurred due to the diffusion of cytologicning; (b) laboratory techniques have improved




Table

All

AbruzzTotal sTotal HHPV ty

1618

16 or 116 or 1Multip

CampaTotal sTotal HHPV ty

1618


LazioTotal sTotal HHPV ty

1618


SardinTotal sTotal HHPV ty

1618


SicilyTotal sTotal HHPV ty

1618



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4. HPV16 and H b pe n gio

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cebp.aloaded from

PV18 identified

5

0.0 7

0.0 60.0 3

0.0 0

8.8 0

5.0 05.0 2

5.0 5

00 0

2.8 58.6 5

0.9 2

0.0 9

0.0 80.0 0

0.0 2

8.9 9

2.5 70 02.5 7

62.5 6

(Con

on Facrjournals.org

y grade, histoty

Squcar

57

0.0 44

3.6 344.5 4

4.9 4

4.1 33

0.0 280.0 3

5.0 2

9.1 46

8.2 304.5 4

0.9 5

7.5 2

1.8 10.0 1

5.1 0

0.0 5

7.8 50.0 07.8 5

66.7 5

tinued on the follow

ebruary 15, 2019. ©

of the lesion, a

no

.0

.3

.1

.1

.7

.8

.1

.1

.9

.2

.7

.9

.0

.0

.0

.0

.0

.0

.0

.0100.0

ing page)

Cancer Epidemiol Bio

2010 American As

d geographic re

ma

22

0 26

3.6 016.4 11

5.5 19

0.0 73

598

12

5.5 20

1.4 474.8 18

9.5 28

0.0 45

6.7 320.0 1

0.0 2

0.0 22

170

1716

markers Prev; 19(9) Se

sociation for Cancer

n

CIN2
CIN3 amous Ade carcino Total
0.0

0.28.7

5.1

8.9

0.81.0

6.4

8.7

6.88.2

2.7

7.8

1.12.2

4.4

5.7

7.30.07.3

72.7

ptember 20

Research.

n
% %
cinoma

%
% % n n n n
144
38 1 36 7
o
ample 4 67 45 11 127 PV+ 4 10 6 10 100 11 10 1 10 pe
4
10 5 8 77 7 6 1 8 0 9 4 3
8 4
10 5 8 84 8 7 1 8 0.0 9 8.1 37 .1 2.7 08 5.78 alone 4 100.0 52 77.6 36 81.8 5 45.5 97 77.0 le infections 0 1 1 9 5 4 1
nia
ample 41 37 46 0 124 PV+ 20 4 2 5 71 0 10 5 pe
11
5 2 10 84 8 3 1 1 9 1
8 12
6 2 10 87 8 0.0 0 0.0 29 .9 61 3.68 alone 9 45.0 15 75.0 29 87.9 53 72.6 le infections 5 2 2 6 1
ample 43 111 47 22 223
PV+ 43 1 11 9 97 21 9 2 9 pe
27
6 7 6 65 15 7 1 6 8 1 8 1
8 33
7 7 7 73 15 7 1 7 6.7 9 1.8 34 .9 1.4 61 3.28 alone 26 60.5 70 63.6 30 65.2 14 66.7 140 63.6 le infections 9 2 1 1 10 2 1
ia
ample 1 40 2 3 46 PV+ 1 10 3 9 100 3 10 9 pe
1
10 2 7 50 2 6 7 0 50 0
8 1
10 2 7 100 2 6 7 0.0 8 1.8 2 .0 6.7 33 3.38 alone 1 100.0 27 69.2 2 100.0 2 66.7 32 71.1 le infections 0 0 0
ample 9 9 5 0 0.0 23
PV+ 8 8 10 100 0 9 pe
5
6 7 100 7 0 0
8 5
6 7 100 7 8 alone 5 le infections 0 0.0 1 11.1 0 0.0 1 4.5
10 2397


specifknowand (cIn I

inviteing. Tther ascreendo Patablishtests atest coNatioern re24% owheremore,Abruzthat Pnon-Htime agroupcovermoreis conHPV1[Westfusionincom13). Tings acollabascribduresmon ttypesof HPFin

laborafor H

ThisresultNapleThe

our dcancecant.in fachighe>80%we obquenccounthavean ovcouldcouldtion oThe

changsive cwithpubliof thefectioproceopinging ccloserThe

der 35portiogroup10-yewomaactivisuggeIn o

Table 4. HPV16 and H b e nd ion

Squ no ma

TuscaTotal sTotal H 7.8 1 0.0 12 .0 0.0 78 .4HPV ty

16 .0 1 .9 9 .0 08 .718 .9 0 .3 1 .3 15 .4

16 o16 oMultiple infections 7 15.6 27 22.3 0 0.0 0 34 19.1

Carozzi et al.

Cance2398

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ically for the most frequent HPV types (we do notwhether bias has been introduced or corrected);) laboratory contamination.taly, public screening programs should activelyall women ages 25 to 64 every 3 years for Pap test-he Pap test, the eventual colposcopy, and any fur-nalysis and treatment are free. Opportunisticing is also widespread, and currently, most womenp test yearly (43). Screening programs are well es-ed in central Italy, whereas in the south most Papre done in opportunistic screening. The 3-year Papverage is high in central Italy [∼80%, according tonal Health Interview (43)] but much lower in south-gions (slightly more than 50%). In our samples, onlyf informative cases are from southern regions,as 76% of them are from central regions. Further-about half of the cases from the south are fromzo, where Pap test coverage is ∼70%. It is possibleap test screening results in better prevention forPV16/18 cancers due to a longer transformationnd to a lower proportion of adenocarcinoma in this(44). Consequently, in a context of high Pap test

age, lesions undetected by screening controls arelikely to be HPV16 or HPV18. This interpretationsistent with the observation that the proportion of6/18 in cancers is higher in high-income countriesern Europe (45) and North America] with large dif-of the Pap test and lower in low/middle- and low-e countries (South America, Asia, and Africa; ref.his hypothesis is in contrast with the reported find-nd with the interpretation given by Wheeler andorators (46). Another plausible explanation mighte this differences to changes in laboratory proce-that result in a higher sensitivity for the most com-ypes and in particular for the two vaccine-targeted(e.g., amplification with the E6/E7-specific primersV16, HPV18, HPV31, and HPV45).ally, contaminations might have occurred during
tory analysis, artificially increasing the positivityPV16, even if all the controls have been done.
in higtrend



hypothesis cannot explain the similarity of thes obtained in different laboratories (Florence ands).proportion of HPV16/18 in adenocarcinoma, in

ata set, is lower than the proportion in squamousrs even if the difference is not statistically signifi-This finding is not consistent with previous results;t, the proportion of HPV18 in adenocarcinomas isr than 35%, and the role of HPV16 + HPV18 isin the large IARC meta-analysis (47). Conversely,served that HPV45 was present with greater fre-y than HPV18 in adenocarcinoma, together ac-ing for 33% of cases. In our data set, we do notany adenocarcinoma in situ and we cannot excludeerstaging of our cases (i.e., some in situ lesionshave been classified as invasive). Overstagingalso explain partially why we have a lower propor-f HPV16/18 in adenocarcinoma.proportion of CIN3 due to HPV16/18 does note with age. On the contrary, the proportion of inva-ancers due to HPV16/18 shows a decreasing trendage. This trend is significant and consistent withshed data (22, 48). This could be the consequencehigher probability of progression in the HPV16 in-n: Higher probability of progression in a multistepss should produce a higher proportion of fast devel-cancers. If this is true, only relatively fast develop-ancer would occur, in younger women, at agesto the start of sexual activity.proportion of non-HPV16/18 among women un-is 8% (1 of 12) in our study. If we apply this pro-n to the very low incidence of cancer in this age(10-year cumulative risk, 50/100,000), the absolute

ar cumulative risk of cervical cancer for an Italiann, ages 25 and vaccinated before the onset of sexualty, will be ∼4/100,000. Similar conclusions werested by Wheeler and collaborators (46).ur data set, the percentage of coinfection decreases

PV18 identified
y grade, histotyp of the lesion, a
her grades; this eis not significant

Cancer Epidem

010 American As

geographic reg

ffect is independentif we adjust for type

iology, Biomarkers &

sociation for Cancer

(Cont'd)

CIN2
CIN3 amous Ade carcino Total
of age. Thof specime

Preventi

Research.

carcinoma

n % n % n % n % n %

ny
ample 46 121 12 0 179 PV+ 45 9 12 10 100 0 1 99 pe
18
40 8 66 75 1 60 4 8 1 8 8 8
8 22
48 8 71 1 83 0 1 66 r 1 .9 7 .9 0 .3 19 .9r 18 alone 17 37.8 74 61.2 10 83.3 0 101 56.7
isn.

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t, there is a higher proportion of coinfections ingic specimens, which in our study were onlyfor CIN2 and CIN3. On the other hand, the trendnificant if we include data on coinfections inhy women (27.7% in the general population,in CIN2, 15.5% in CIN3, and 10.0% in invasive

rs; ref. 49). In the literature, there are inconsistentusions about this point (50, 51) even if moret studies seem to be consistent with our data0, 24).ee possible mechanisms may explain this finding:nfections by types other than HPV16 could be pro-e for progression; (b) the progression could cause aexpansion of the infection, reducing the probabili-detect a coinfection; and (c) if the percentage oflogic tissue in samples of invasive cancers is higherthe surrounding healthy tissue, the probability offying noncancerous infections in other parts of thecould be reduced.e first explanation is true, the effect of vaccinatione difficult to predict. On the other hand, it is diffi-r retrospective studies like ours to predict the effectcination in preventing CIN2 and CIN3 sustained
infection because it is impossible to know which
Discl

F.M.and Gl

Ackn

Wespecim

Grant

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type causes the lesion.

lusions

proportion of invasive cancer with HPV16/18ntral and southern Italy is 78%, and althoughinding is consistent with previous results, itd be emphasized that it represents the highest.proportion of invasive cancers due to HPV16/18ses with age at diagnosis. The absolute risk of anve cancer due to non-HPV16/18 in women underextremely low and probably does not justify any
ing among younger women if they have been vac- Rece
de 3 and cancer: 5-year follow-up of a randomised controlledlementation trial. Lancet 2007;24:1764–72.ucler P, Ryd W, Törnberg S, et al. Human papillomavirus and

Pa20

7. Kitbin(AR67

8. MaCaverMe

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10. CliJMcece11

Cacrjournals.org


ndix:HPVPrevalence ItalianWorkingGroup

zo: Angeloni Claudio, Lattanzi Amedeo, Maccallininzo, Caraceni Donatella, Fortunato C. Cagliari:Rosalba, PiliaMassimo, CareddaValeria. Campania:o Giuseppe, Di Iasi Angela, Santarsiere Aldo, Castoana, Manno Maria, Santangelo Claudia, Pini Maria, Gallicchio Giuseppina, Scherillo Isabella, Barretta, De Santis Vincenzo, Ercole Filomena. Catania:i Aurora, Spampinato G., Cantarella M.A., MianoLazio: Giorgi Rossi Paolo, Chini Francesco,arucci Paola, Marsili Laila Maria, Tufi Mariatta, Gomez Vito, Verrico Giovanna, Schiboni Maria, Pellegrini Antonella, Bove Emilia, D'Addettaa, Placidi Antonio. National Cancer Institute Fondle: Buonaguro Franco Maria, Tornesello Maria Lina,ercio Giovanna, Losito Simona, Botti Gerardo,ioneAldo.Tuscany:Carozzi FrancescaM., Confortiniimo, Bisanzi Simonetta, Sani Cristina, Venturini, Burroni Elena, Tinacci Galliano.Ministry ofHealth:io Federici.

osure of Potential Conflicts of Interest

Carozzi is an occasional advisor to Gen-Probe, Abbott, Sanofi,axoSmithKline.

owledgments

thank all the pathology unit staff involved in the retrieving ofens and Giovanna Patrone for the English editing.

Support

study has been financed by the Italian Ministry of Health and byticipating centers.costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedement in accordance with 18 U.S.C. Section 1734 solely to indicatet.

ived 02/05/2010; revised 06/20/2010; accepted 06/25/2010;
d before the onset of sexu al activity. published online 09/08/2010.
rencesnley M. Prevention strategies against the human papillomavi-: the effectiveness of vaccination. Gynecol Oncol 2007;107:9–23.tts FT, Franceschi S, Goldie S, et al. Human papillomavirus andV vaccines: a review. Bull World Health Organ 2007;85:719–26.zick J, Clavel C, Petry KU, et al. Overview of the European andrth American studies on HPV testing in primary cervical cancereening. Int J Cancer 2006;119:1095–101.nco G, Giorgi-Rossi P, Carozzi F, et al, New Technologies forrvical Cancer Screening Working Group. Human papillomavirusting and liquid-based cytology in primary screening of womenunger than 35 years: results at recruitment for a randomisedntrolled trial. Lancet Oncol 2006;7:547–55.lkmans N, Berkhof J, Rozendaal L, et al. Human papillomavirusA testing for the detection of cervical intraepithelial neoplasia

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ancer Epidemiol Biomarkers Prev; 19(9) September 2010 2399



11. DeEu

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2010;19:2389-2400. Cancer Epidemiol Biomarkers Prev Francesca M. Carozzi, Maria L. Tornesello, Elena Burroni, et al. Cervical Intraepithelial Neoplasia and Cancer in ItalyPrevalence of Human Papillomavirus Types in High-Grade

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